CA1331017C - Process for the seperation of 2-ceto-l-gulonic acid from a fermentation wort - Google Patents

Abstract

Process for separating 2-keto-L-gulonic acid from a fermentation liquor chiefly containing the calcium salt of 2-keto-L-gulonic acid, by carrying out the following successive operations: 1) separation of the insoluble matter, 2) demineralisation of the filtered liquor, and 3) separation of the 2-keto-L-gulonic acid.

Description

- 133 ~ 017 2-kulose gulonic acid, an intermediate in the synthesis of ascorblque acid, is generally present in musts of iron ~ entation of appropriate microorganisms under the form of keto-2 L
calcium yulonate.
For 9 ~ transformation into ascorbic acid, it is partl-especially advantageous to be able to deposit 2-keto acid gulonic either 80US free form (2XLG-H) or 80US salt form sodium (2XLG-Na) 90US as pure as possible.
According to patent applications ~ aponais JP 52.66684 and 10 JP 53.62894, it is known to prep ~ r the keto-2 L sodium gulonate practically pure from a fermentation must containing 2-keto calcium gulonate. However, the industrial implementation trielle of these processes does not allow to directly obtain the acid keto-2 L gulonic necessary for subsequent trans ~ ormation in ascorbic acid.
It has now been found, and it is ~ e which makes the ob ~ and of the present invention, that 2-kulo gulonic acid (2XLG-H) practically pure can be obtained with good yields from fermentation musts containing 2-keto calcium gulonate by a simple and easily reproducible process.
The fermentation media leading to 2XLG-H contain-nent generally, next to the Gcide 2-keto gulonic form of ~ el of calcium, an insoluble biomass and organic impurities nlques or mineral, mineral impurities conslst ~ nt essential-inorganic anions associated with metal cations such as than sodium, potassium or magnesium ions.
The method according to the invention essentially consists of carrying out the following successive steps:
1) separation of the insoluble matter from the fermenting must,

2) demineralization of the filtered mo ~ t,

3) separation of the gulonic keto-2 L cide.
The implementation of the process can be carried out continuously or in discontlnu.
Biomass and insoluble substances which represent generally 1.5 to 3% by weight of the whole must may be ~ spared from the mo ~ t:

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~ ~ 331 ~ 17 - either by centrlfugatlon ~ near flocculation in the kernel of a flocculating agent such as, for example, of polyacrylamide type, - or by filtratlsn under reduced pressure after ~ floccu-lation by means of a ~ flocculating gent such as, for example the poly-acryla ~ lde, and additlon ad ~ uvant of flltr ~ tion such as, for example, the farlne of bowls or the earth of dlatomées, - either by ultrafiltration through mineral membranes r ~ the ~ or appropriate organic such as the ~ polyvinyl membranes difluorées or uembrane ~ constltuelles of ZrO2 on matrlce of carbon.
The ~ got rid of biomass and ~ substances insoluble can be concentrated, solt by ~ vaporatlon under presslon reduced to a temp ~ rature lnférleure at 60C ~ until a volume corresponding between 1/4 and 1 ~ 3 of the initial volume, either by osmosis reverse on a poly membrane ~ ulfonée ~ a temperature close to 50C ~ until the noltl ~ around its volume.
The concentrated must is acidified by addition concentrated sulfuric acid in practically stoichiometric quantities trlque by r ~ report to the calclum present ~ a lower temperature or ~ scab at 40C. The precluded calclum sulfate is separated by flltratlon and is washed with water. The filter that contains catlons dlver ~ whose prlnclpnux are the calcluu not preclpit ~, the sodlum, the pot ~ s ~ ium and the magn ~ sium is decatlonlsé and acidified p ~ r passage on a column of r ~ slne polycyclic cation exchange cycle acid, preferably of the sulfonic type.
It is also possible, before concentration, to decatlonlser and acidlfler the noût p ~ r passage dlrect on a réslne cationlque under for ~ e aclde, preferably type sulfonlque.
The start no ~ rrass ~ of the sedlnent ~ ot of the catlons, possibly-lement concentr ~, is then freed of the anions in solution by pass ~ ge nur una ré ~ lno polymérlque ~ chanter of anlons, pref ~ -dlalkylanlno type rence.
The ~ olutlon denln ~ rall ~ ée can then be concentrated by pressure evaporation r ~ following a temperature ~ lower temperature ~ lower ~ 60C

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3 ~ 331 ~ 17 ~ up to ~ 70% of the initial volume. The crystallization of the gulonic keto-2 L coincide is obtained by a concentration 8upple ~
under reduced pressure at a temperature close to 40C
~ until a decrease in volume co ~ taken between 30 and 40% approximately possibly followed by cooling of the boulllie crystalline.
It may be particularly advantageous to perform the continuous crystallization in a crystallizer operating by evaporation under reduced pressure at a voline temperature of 40C and fitted with an external exchanger. The crystals obtained are separated by filtration or by pulsing puls washed with water. The aclde 2-keto gulonic is isolated in the form of a monohydrate, the purity is generally close to 100% and whose water molecule hydration can be removed by heating under pressure scaled down.
Usually drying of 2-keto acid crystals gulonic monohydrate wet is carried out by transport under a flow hot air.
According to the present invention, 2-kulose gulonic acid can also be obtained after extraction of concentrated and demineralized must produced using a suitable organic solvent chosen from optionally halogenated aliphatic or aromatic hydrocarbons containing in solution an aliphatic amine, preferably second-daire, containing more than 20 carbon atoms. The organic solution thus obtained can be extracted with an aqueous solution of an acid strong mineral chosen from hydrochloric, sulfuric or nitric whose concentration can be between 0.5N and 3N.
The aqueous solution thus obtained can be concentrated dry to lead to a 2-keto acid ~ ulonic monohydrate under pulverulent form, the purity of which is generally greater than 90%.
The following examples, given without limitation, show how the invention can be put into practice.

4 1 3 3 1 0 1 7 B EMPLE 1 - SéDarstiOn of insoluble must from fermentation We use a fermentation must obtained by culture a strain of corynebactérlum producing keto-2 L acid gulo-nlque containing about 1.6% of biomass and lnsolu- substances wheat and 10% keto-2 L calcium gulonate.
Blomass and insoluble substances can be separated by one of the following methods:
1) We must pass 123 kg of fermenting must containing about 2 kg of sediment through an ultraflltratlon membrane 10 CARBOSEP with a porosity of 80,000 DA, with a temperature of 40C. This gives 112 kg of permeate free of lnso-lubles and 11 kg of retentate containing all of the seeds and in which the concentration of 2-keto calclum gulonate is ~ equal to that in the permeate.
The retentate is exhausted by additive diafiltration ¦ successive water at the retentate during an identical ultrafiltration ¦ tlque ~ that described pr ~ c ~ demment. Diaflltration is stopped when the content of 2-keto calcium gulonate in the combined permeates corresponds to a recovery rate of at least 99%.
~ 20 2) To 500 g of whole must, vigorously stirred, we add ¦ 20 cm3 of an aqueous solution ~ 5 g / liter of flocculant of the type polyacryla ~ ide FLOERGER 8850. After a few minutes of contact, we in addition, while maintaining vigorous agitation, 10 9 of fGrine of wood.
The ~ mixture obtained is filtered on a filter cloth under a I reduced pressure (300 mm nercure; 40 kPa).
c The filter cake is washed with 50 cm3 of water.
The loss of c ~ to-2 L culium gulonate is less than 2 ~.
, 30 The weight of the filtrate contains 9.4% of c ~ to-2 L gulonate ¦ of calcium is da 520 g.
3) 200 lltre ~ of whole mo containing t ~ 2,7 ~ of s ~ dl ~ ents are flocculated with 8 liters of an aqueous solution ~ 5 9 /
liter of a polyacrylamide type fluxing agent (ELOERGE ~ 8850) then are introduced continuously at a flow rate of 2000 liters / hour into i * trademarks : ~ ,,, ~., .. -. ,,. :, ~.

1331 ~ 7 a centrifugal clarlficatrlce with plates of 7200 n2 of surface - equivalent.
We collected a clarified mo ~ t containing 0.1% of seductive.

EXAMPLE 2 Concentration of the must The wort released from the soluble can be concentrated according to one of the ~ su ~ vantes methods:
l) Concentrate 225 liters of must which is insoluble in evaporatlon under pres ~ lon reduced (72 nm of mercury; 9.5 kP ~) to a temperature of 47C ~ up to a volume of 62 liters.
The degradatlon ther ~ lque is lnférleure at 0.5%.
2) 60 liters of insoluble must must are concentrated by passage ~ through a lnverse osmosis module fitted with membranes polysulfonated (PCI Z 99) at a flow rate of 25 lltrec / h.m2 and ~ one temp ~ rature less than or equal to 50C ~ usqu '~ obtlr a volume of 30 liters.

EXAMPLE 3 Precipitation of calcium sulphate In a stirred reactor, maintained at a temperature below is equal to or equal to ~ 40C and containing 2 liters of concentrated must obtained in the condltions decltes in example 2-l, we have ~ oute a quantity of concentrated sulfuric acid corresponding, in moles, to the total quantity of calclum present in the reactor is l00 cm3.
The calcium sulphate which precludes in the form of dlhy-drate is separated by filtration and washed with water.
The combined filtrate and washes contlenn ~ nt 99.5%, mole ~, c ~ to-2 L gulonic acid initlal.
The yield of calclum removal is 95%.
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EXAMPLE 4 - Elimination of cations We pass 3 liters of the flltrate obtained in Example 3 (before to be mixed nux wash water from calclum sulfate cake) on a column 80 cm high and 5 cm in diameter constant 1.6 liters of strong cationic resin (A ~ berlite * IRC 120) in cycle acid.

* trademark i:. ~. . . , -. ,, After washing, we obtain 5.5 liters of mo ~ t in which the sulfuric ash content is less than 1% by rspport ~ 2-kulo gulonic acid present in the solution.
The t ~ ux of recovery of 2-keto acid gulonic is higher ~ 99.5%.

EXAMPLE 5 Elimination of anions 0.4 liter of the must obtained in Example 4 is passed through through a column 10 cm high and 1.4 cm in diameter 15 cm3 of weak anionic reslne ~ Amberlite IRA 93) in order to reduce the sulfuric acid content in the must by a factor of 10 acidifle.
The loss of 2-kulose gulonic acid does not exceed ~% of the quantity entered.

EXAMPLE 6 - Crystallization of 2-kulose gulonic acid 5000 9 of ~ cost obtained under the conditions of Example 5 are concentrated by evaporation so as to obtain 3430 9 of a solution containing 1170 9 2-keto-gulonic acid and 255 9 impurities.
The concentrate thus obtained is partially concentrated under reduced pressure (40 mm Hg; 5.3 kPa) at a temperature from 40C up to a weight of 2010 9. This concentration supplements shut up leads to the crystallization of 2 L gulonic keto acid hydr ~ tee. The crystal slurry is cooled to 25C. Crystals are separated by filtration and washed with water. We thus obtain 930 9 hydrated 2-keto gulonic acid of higher purity 99% and whose sulfuric ash content is less than 0.1%.
The yield is 73%.
Filtration mother liquors and washing water combined then concentrated under reduced pressure (40 mm of nercure

5.3 kPa) up to a weight of 710 g. After cooling, the 80nt crystals separated by filtration and washed with water. We obtain thus 285 9 of ~ keto-2 L gulonic monohydr ~ ty whose purity is 89%.

13310 ~ 7 -Crystals of 2-keto acid Gulonic ~ onohydrate wet (5.8% water) with a purity greater than 99% (9400 g) are dried by air at 75C circulating at 5 m / s in a dryer pneumatic transport, the residence time being 3 seconds.
8850 g of keto-2 L gulonic mono-hydrate. The ~ 2-keto clul gulonlque uohohydrate can be dehydrated by heating to 40C under reduced pressure (5 mm of mercury 0.67 kPa) for several hours.

One puts in contact 1 liter of must freed from sedl ~ ents and cations containing 80% of 2-keto-gulonic acid having a 84% purity with 1 liter of 260 9 AWGEN 83 solution (Registered trademark SCHERING) in kerosene for 0.5 hour at 50C.
The 2-kulose gulonic acid (83%) which is extracted in the organic phase is re-extracted quantitatively by 690 cm3 of a 1N aqueous nitric acid solution.
The concentrated dry solution provides 81.5 g of a product crystallized containing 89% 2-keto-gulonic acid monohydrate.
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Claims (7)

1. Process for obtaining 2-keto acid gulonic from a fermentation must containing calcium salt of this 2-ketulose gulonic acid and insoluble substances, characterized in that:
- the insoluble substances contained are separated in the must;
- demineralize the must, which has been removed from its insoluble substances by acidification with acid sulfuric added in stoichiometric quantity compared with calcium present in the must, then separation of the sulfate of calcium precipitated by filtration, and finally passage of the filtrate first on a cation exchange column in acid cycle, then on an anion exchange column; and - separating 2-kulose gulonic acid from said must demineralized. 2. Method according to claim 1, characterized in that the insoluble substances contained in the must fermentation are separated;
either by centrifugation, after flocculation using a flocculating agent;
either by filtration after flocculation by means of a flocculating agent and addition of a filter aid;
either by ultra-filtration. 3. Method according to claim 2, characterized in that, after the separation of the insoluble substances, the must is concentrated before being demineralized. 4. Method according to claim 1, 2 or 3, characterized in that the demineralized must is concentrated and in that 2-kulo gulonic acid is separated by crystallization followed by filtration. 5. Method according to claim 1, 2 or 3, characterized in that the 2-kulo gulonic acid is separated concentrated and demineralized must. by extraction using of an organic solvent chosen from hydrocarbons phatic or aromatic halogenated or not containing in solution an aliphatic amine having more than 20 atoms of carbon, then re-extraction using an aqueous solution a strong mineral acid chosen from chlorhy-dric, nitric and sulfuric, and dry concentration of aqueous solution obtained. 6. Method according to claim 1, 2 or 3, characterized in that it is implemented continuously. 7. Method according to claim 1, 2 or 3, characterized in that it is implemented discontinuously.